Pentose sugar fermenting cell

ABSTRACT

The invention relates to a cell which comprises a nucleotide sequence encoding a xylose isomerase, wherein the amino acid sequence of the xylose isomerase has at least 75% sequence identity to the amino acid sequence set out in SEQ ID NO: 2 and wherein the nucleotide sequence is heterologous to the host. A cell of the invention may be used in a process for producing a fermentation product, such as ethanol. Such a process may comprise fermenting a medium containing a source of xylose with a cell of the invention such that the cell ferments xylose to the fermentation product.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional Application of U.S. patent applicationSer. No. 14/236,191, filed Jan. 30, 2014, which is a National PhaseApplication of PCT/EP2012/065088, filed Aug. 2, 2012, which claimspriority to European Application No. 11176601.0, filed Aug. 4, 2011, andto U.S. Provisional Application No. 61/515,014, filed Aug. 4, 2011.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to a cell which is capable of isomerisingxylose to xylulose. The invention also relates to a process in whichsuch cells are used for the production of a fermentation product, suchas ethanol.

Description of Related Art

Large-scale consumption of traditional, fossil fuels (petroleum-basedfuels) in recent decades has contributed to high levels of pollution.This, along with the realisation that the world stock of fossil fuels isnot limited and a growing environmental awareness, has stimulated newinitiatives to investigate the feasibility of alternative fuels such asethanol, which is a particulate-free burning fuel source that releasesless CO₂ than unleaded gasoline on a per liter basis.

Although biomass-derived ethanol may be produced by the fermentation ofhexose sugars obtained from many different sources, the substratestypically used for commercial scale production of fuel alcohol, such ascane sugar and corn starch, are expensive. Increases in the productionof fuel ethanol will therefore require the use of lower-cost feedstocks.

Currently, only lignocellulosic feedstock derived from plant biomass isavailable in sufficient quantities to substitute the crops currentlyused for ethanol production. In most lignocellulosic material, thesecond-most-common sugar, after glucose, is xylose. Thus, for aneconomically feasible fuel production process, both hexose and pentosesugars must be fermented to form ethanol. The yeast Saccharomycescerevisiae is robust and well adapted for ethanol production, but it isunable to produce ethanol using xylose as a carbon source. Also, nonaturally-occurring organisms are known which can ferment xylose toethanol with both a high ethanol yield and a high ethanol productivity.There is therefore a need for an organism possessing these properties soas to enable the commercially-viable production of ethanol fromlignocellulosic feedstocks.

SUMMARY

According to the invention, there is provided a cell that is capable offermentation, such as alcoholic fermentation, and of using xylose as acarbon source. Such a cell comprises a nucleotide sequence encoding axylose isomerase, wherein the amino acid sequence of the xyloseisomerase has at least 75% sequence identity to the amino acid sequenceset out in SEQ ID NO: 2 and wherein the nucleotide sequence isheterologous to the host. Such a cell produces a higher amount ofethanol when using xylose as a carbon source as compared to the wildtype filamentous fungus.

The invention also provides:

-   -   a process for producing a fermentation product which process        comprises fermenting a medium containing a source of xylose with        a cell of the invention such that the cell ferments xylose to        the fermentation product;

a process for producing a fermentation product which process comprisesfermenting a medium containing at least a source of xylose and a sourceof L-arabinose with a cell as defined of the invention which is alsocapable of utilizing L-arabinose such that the cell ferments xylose andL-arabinose to the fermentation product; and

-   -   a process for producing a fermentation product which process        comprises fermenting a medium containing at least a source of        xylose and a source of L-arabinose with a cell of the invention        and a cell able to use L-arabinose, whereby each cell ferments        xylose and/or arabinose to the fermentation product.

In an embodiment, the cell comprises a nucleotide sequence encoding axylose isomerase obtainable from a cell of genus Clostridium, e.g. froma Clostridium beijerinckii cell. In an embodiment, the nucleotide may bewild-type or codon optimized or codon pair optimized.

The invention further provides the use of a cell of the invention in aprocess for the production of a fermentation product.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 sets out the plasmid map of p427-TEF encoding xylose isomerasefrom Clostridium beijerinckii for expression in Saccharomycescerevisiae. CpO denotes codon pair optimized.

FIG. 2 sets out a growth curve of BIE104P1 transformed with pPWT215 on2% xylose as sole carbon source, and of the reference strain transformedwith no DNA in the plasmid (mock transformation). The numbers “1” and“2” indicate the transfer of an aliquot of the culture to fresh medium.All cultures were incubated in the presence of air, except the last one(after the second transfer).

FIG. 3 sets out growth curves of Strain BIE292XI (C. beyerinckii) (blackline) and a mock strain (grey line), from example 6.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 sets out the codon pair optimised xylose isomerase sequencefrom Clostridium beijerinckii.

SEQ ID NO: 2 sets out the amino acid sequence of xylose isomerase fromClostridium beijerinckii.

SEQ ID NO: 3 sets out the sequence of forward primer.

SEQ ID NO: 4 sets out the sequence of reverse primer.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

Throughout the present specification and the accompanying claims thewords “comprise” and “include” and variations such as “comprises”,“comprising”, “includes” and “including” are to be interpretedinclusively. That is, these words are intended to convey the possibleinclusion of other elements or integers not specifically recited, wherethe context allows.

The invention relates to a cell which comprises a nucleotide sequenceencoding a xylose isomerase, wherein the amino acid sequence of thexylose isomerase has at least 75% identity to the amino acid sequenceset out in SEQ ID NO: 2 and wherein the nucleotide sequence isheterologous to the host.

The presence of the nucleotide sequence encoding a xylose isomeraseconfers on the cell the ability to isomerise xylose to xylulose.

A “xylose isomerase” (EC 5.3.1.5) is herein defined as an enzyme thatcatalyses the direct isomerisation of D-xylose into D-xylulose and/orvice versa. The enzyme is also known as a D-xylose ketoisomerase. Axylose isomerase herein may also be capable of catalysing the conversionbetween D-glucose and D-fructose (and accordingly may therefore bereferred to as a glucose isomerase). A xylose isomerase herein mayrequire a bivalent cation, such as magnesium, manganese or cobalt as acofactor.

Accordingly, a cell of the invention is capable of isomerising xylose toxylulose. The ability of isomerising xylose to xylulose is conferred onthe host cell by transformation of the host cell with a nucleic acidconstruct comprising a nucleotide sequence encoding a defined xyloseisomerase. A cell of the invention isomerises xylose into xylulose bythe direct isomerisation of xylose to xylulose. This is understood tomean that xylose is isomerised into xylulose in a single reactioncatalysed by a xylose isomerase, as opposed to two step conversion ofxylose into xylulose via a xylitol intermediate as catalysed by xylosereductase and xylitol dehydrogenase, respectively.

A unit (U) of xylose isomerase activity may herein be defined as theamount of enzyme producing 1 nmol of xylulose per minute, underconditions as described by Kuyper et al (2003, FEMS Yeast Res. 4:69-78).

The cell of the invention is defined with reference to a xyloseisomerase having the amino acid sequence of SEQ ID NO: 2 or a sequencehaving at least 74% sequence identity thereto. Likewise, a cell of theinvention may be defined with reference to a xylose isomerase be anucleotide sequence which encoding such an amino acid sequence.

SEQ ID NO: 2 sets out the amino acid sequence of xylose isomerase fromClostridium beijerinckii. A cell of the invention comprises a nucleotidesequence encoding a xylose isomerase having the amino acid of SEQ ID NO:2 or one which has at least 74% sequence identity thereto.

Preferably, a cell according to the present invention is a cellcomprising a nucleotide sequence encoding a xylose isomerase having asequence which has at least about 75%, preferably at least about 80%, atleast about 85%, at least about 90%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, atleast about 97%, at least about 98% or at least about 99% or at least75%, at least 80%, at least 85%, at least 90%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98% or at least 99% sequence identity with the amino acid sequence ofSEQ ID NO:2. A cell according to the present invention may comprise anucleotide sequence encoding a xylose isomerase having a sequence whichhas at least about 50%, at least about 55%, at least about 60%, at leastabout 70%, at least about 75%, preferably at least about 80%, at leastabout 85%, at least about 90%, at least about 92%, at least about 93%,at least about 94%, at least about 95%, at least about 96%, at leastabout 97%, at least about 98% or at least about 99% or at least 75%, atleast 80%, at least 85%, at least 90%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98% or atleast 99% sequence identity with the nucleic acid sequence set out inSEQ ID NO: 1.

Sequence identity (or sequence similarity) is herein defined as arelationship between two or more amino acid (polypeptide or protein)sequences or two or more nucleic acid (polynucleotide) sequences, asdetermined by comparing the sequences. Usually, sequence identities orsimilarities are compared, typically over the whole length of thesequences compared. However, sequences may be compared over shortercomparison windows. In the art, “identity” also means the degree ofsequence relatedness between amino acid or nucleic acid sequences, asthe case may be, as determined by the match between strings of suchsequences.

Preferred methods to determine identity are designed to give the largestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Preferred computer program methods to determine identity and similaritybetween two sequences include e.g. the BestFit, BLASTP, BLASTN, andFASTA (Altschul, S. F. et al., J. Mol. Biol. 215:403-410 (1990),publicly available from NCBI and other sources (BLAST Manual, Altschul,S., et al., NCBI NLM NIH Bethesda, M.D. 20894). Preferred parameters foramino acid sequences comparison using BLASTP are gap open 11.0, gapextend 1, Blosum 62 matrix. Preferred parameters for nucleic acidsequences comparison using BLASTP are gap open 11.0, gap extend 1, DNAfull matrix (DNA identity matrix).

Optionally, in determining the degree of amino acid similarity, theskilled person may also take into account so-called “conservative” aminoacid substitutions, as will be clear to the skilled person.

Conservative amino acid substitutions refer to the interchangeability ofresidues having similar side chains. For example, a group of amino acidshaving aliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulphur-containing sidechains is cysteine and methionine.

Preferred conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, and asparagine-glutamine. Substitutional variants of theamino acid sequence disclosed herein are those in which at least oneresidue in the disclosed sequences has been removed and a differentresidue inserted in its place. Preferably, the amino acid change isconservative. Preferred conservative substitutions for each of thenaturally occurring amino acids are as follows: Ala to ser; Arg to lys;Asn to gln or his; Asp to glu; Cys to ser or ala; Gin to asn; Glu toasp; Gly to pro; His to asn or gln; He to leu or val; Leu to ile or val;Lys to arg; gln or glu; Met to leu or ile; Phe to met, leu or tyr; Serto thr; Thr to ser; Trp to tyr; Tyr to trp or phe; and, Val to ile orleu.

A nucleotide sequence encoding an enzyme which catalyses the conversionof xylose to xylulose according to the invention may also be defined byits capability to hybridise with the nucleotide sequences encoding theenzyme having the sequence set out in SEQ ID NO: 2 or a sequence havingat least 74% sequence identity therewith, under moderate, or preferablyunder stringent hybridisation conditions.

Formally, such nucleotide sequences hybridize with the reversecomplement of the nucleotide sequences which encode the enzyme havingthe sequence set out in SEQ ID NO: 2 or a sequence having at least 74%sequence identity therewith, for examples sequences which hybridize withthe reverse complement of SEQ ID NOs: 1 or 2.

Stringent hybridisation conditions are herein defined as conditions thatallow a nucleic acid sequence of at least about 25, preferably about 50nucleotides, 75 or 100 and most preferably of about 200 or morenucleotides, to hybridise at a temperature of about 65° C. in a solutioncomprising about 1 M salt, preferably 6×SSC (sodium chloride, sodiumcitrate) or any other solution having a comparable ionic strength, andwashing at 65° C. in a solution comprising about 0.1 M salt, or less,preferably 0.2×SSC or any other solution having a comparable ionicstrength. Preferably, the hybridisation is performed overnight, i.e. atleast for 10 hours and preferably washing is performed for at least onehour with at least two changes of the washing solution. These conditionswill usually allow the specific hybridisation of sequences having about90% or more sequence identity.

Moderate conditions are herein defined as conditions that allow anucleic acid sequences of at least 50 nucleotides, preferably of about200 or more nucleotides, to hybridise at a temperature of about 45° C.in a solution comprising about 1 M salt, preferably 6×SSC or any othersolution having a comparable ionic strength, and washing at roomtemperature in a solution comprising about 1 M salt, preferably 6×SSC orany other solution having a comparable ionic strength. Preferably, thehybridisation is performed overnight, i.e. at least for 10 hours, andpreferably washing is performed for at least one hour with at least twochanges of the washing solution. These conditions will usually allow thespecific hybridisation of sequences having up to 50% sequence identity.The person skilled in the art will be able to modify these hybridisationconditions in order to specifically identify sequences varying inidentity between 50% and 90%.

To increase the likelihood that the introduced enzyme is expressed inactive form in a cell of the invention, the corresponding encodingnucleotide sequence may be adapted to optimise its codon usage to thatof the chosen yeast cell. Several methods for codon optimisation areknown in the art. A preferred method to optimise codon usage of thenucleotide sequences to that of the yeast is a codon pair optimizationtechnology as disclosed in WO2006/077258 and/or WO2008/000632.WO2008/000632 addresses codon-pair optimization. Codon-pair optimisationis a method wherein the nucleotide sequences encoding a polypeptide aremodified with respect to their codon-usage, in particular thecodon-pairs that are used, to obtain improved expression of thenucleotide sequence encoding the polypeptide and/or improved productionof the encoded polypeptide. Codon pairs are defined as a set of twosubsequent triplets (codons) in a coding sequence.

As a simple measure for gene expression and translation efficiency,herein, the Codon Adaptation Index (CAI), as described in Xuhua Xia,Evolutionary Bioinformatics 2007: 3 53-58, is used. The index uses areference set of highly expressed genes from a species to assess therelative merits of each codon, and a score for a gene is calculated fromthe frequency of use of all codons in that gene. The index assesses theextent to which selection has been effective in moulding the pattern ofcodon usage. In that respect it is useful for predicting the level ofexpression of a gene, for assessing the adaptation of viral genes totheir hosts, and for making comparisons of codon usage in differentorganisms. The index may also give an approximate indication of thelikely success of heterologous gene expression. In the codon pairoptimized genes according to the invention, the CAI is 0.6 or more, 0.7or more, 0.8 or more, 0.85 or more, 0.87 or more 0.90 or more, 0.95 ormore, or about 1.0.

In a cell of the invention, the xylose isomerase is typicallyheterologous to the cell. That is to say, the xylose isomerase has asequence which does not naturally occur in the cell in question as partof the organism, cell, genome DNA or RNA sequence in which it ispresent. That is to say, the xylose isomerase is exogenous to the cellor does not occur naturally in the cell. Accordingly, a nucleotidesequence encoding a xylose isomerase is typically expressed or iscapable of being expressed in active form in the transformed host cell.

A cell of the invention is thus a cell that comprises, i.e. has beentransformed with, a nucleic acid construct comprising the nucleotidesequence encoding the xylose isomerase as defined above. The nucleicacid construct comprising the xylose isomerase coding sequencepreferably is capable of expression of the xylose isomerase in the hostcell.

Methods for expressing a heterologous xylose isomerase sequence in acell are well known to those skilled in the art.

Accordingly, a cell of the invention is a recombinant cell. That is tosay, a cell of the invention comprises, or is transformed with or isgenetically modified with a nucleotide sequence that does not naturallyoccur in the cell in question.

Techniques for the recombinant expression of xylose isomerase in a cell,as well as for the additional genetic modifications of a cell of theinvention are well known to those skilled in the art. Typically suchtechniques involve transformation of a cell with nucleic acid constructcomprising the relevant sequence. Such methods are, for example, knownfrom standard handbooks, such as Sambrook and Russel (2001) “MolecularCloning: A Laboratory Manual (3rd edition), Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, or F. Ausubel et al.,eds., “Current protocols in molecular biology”, Green Publishing andWiley Interscience, New York (1987). Methods for transformation andgenetic modification of fungal host cells are known from e.g. EP-A-0635574, WO 98/46772, WO 99/60102, WO 00/37671, WO90/14423, EP-A-0481008,EP-A-0635574 and U.S. Pat. No. 6,265,186.

Most episomal or 2μ plasmids are relatively unstable, being lost inapproximately 10⁻² or more cells after each generation. Even underconditions of selective growth, only 60% to 95% of the cells retain theepisomal plasmid. The copy number of most episomal plasmids ranges from10-40 per cell of cir⁺ hosts. However, the plasmids are not equallydistributed among the cells, and there is a high variance in the copynumber per cell in populations. Strains transformed with integrativeplasmids are extremely stable, even in the absence of selectivepressure. However, plasmid loss can occur at approximately 10⁻³ to 10⁻⁴frequencies by homologous recombination between tandemly repeated DNA,leading to looping out of the vector sequence. Preferably, the vectordesign in the case of stable integration is thus, that upon loss of theselection marker genes (which also occurs by intramolecular, homologousrecombination) that looping out of the integrated construct is no longerpossible. Preferably the genes are thus stably integrated. Stableintegration is herein defined as integration into the genome, whereinlooping out of the integrated construct is no longer possible.Preferably selection markers are absent.

Typically, the nucleic acid construct may be a plasmid, for instance alow copy plasmid or a high copy plasmid. The cell according to thepresent invention may comprise a single or multiple copies of thenucleotide sequence encoding a xylose isomerase, for instance bymultiple copies of a nucleotide construct or by use of construct whichhas multiple copies of the xylose isomerase sequence.

The nucleic acid construct may be maintained episomally and thuscomprise a sequence for autonomous replication, such as an autosomalreplication sequence sequence. A suitable episomal nucleic acidconstruct may e.g. be based on the yeast 2μ or pKD1 plasmids (Gleer etal., 1991, Biotechnology 9: 968-975), or the AMA plasmids (Fierro etal., 1995, Curr Genet. 29:482-489). Alternatively, each nucleic acidconstruct may be integrated in one or more copies into the genome of thecell. Integration into the cell's genome may occur at random bynon-homologous recombination but preferably, the nucleic acid constructmay be integrated into the cell's genome by homologous recombination asis well known in the art (see e.g. WO90/14423, EP-A-0481008, EP-A-0635574 and U.S. Pat. No. 6,265,186).

Typically, the xylose isomerase encoding sequence will be operablylinked to one or more nucleic acid sequences, capable of providing foror aiding the transcription and/or translation of the xylose isomerasesequence.

The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. For instance, a promoter or enhancer isoperably linked to a coding sequence the said promoter or enhanceraffects the transcription of the coding sequence.

As used herein, the term “promoter” refers to a nucleic acid fragmentthat functions to control the transcription of one or more genes,located upstream with respect to the direction of transcription of thetranscription initiation site of the gene, and is structurallyidentified by the presence of a binding site for DNA-dependent RNApolymerase, transcription initiation sites and any other DNA sequencesknown to one of skilled in the art. A “constitutive” promoter is apromoter that is active under most environmental and developmentalconditions. An “inducible” promoter is a promoter that is active underenvironmental or developmental regulation.

The promoter that could be used to achieve the expression of anucleotide sequence coding for an enzyme according to the presentinvention, may be not native to the nucleotide sequence coding for theenzyme to be expressed, i.e. a promoter that is heterologous to thenucleotide sequence (coding sequence) to which it is operably linked.The promoter may, however, be homologous, i.e. endogenous, to the hostcell.

Suitable promoters in this context include both constitutive andinducible natural promoters as well as engineered promoters, which arewell known to the person skilled in the art. Suitable promoters ineukaryotic host cells may be GAL7, GAL10, or GAL1, CYC1, HIS3, ADH1,PGL, PH05, GAPDH, ADC1, TRP1, URA3, LEU2, ENO1, TPI1, and AOX1. Othersuitable promoters include PDC1, GPD1, PGK1, TEF1, and TDH3.

In a cell of the invention, the 3′-end of the nucleotide acid sequenceencoding xylose isomerase preferably is operably linked to atranscription terminator sequence. Preferably the terminator sequence isoperable in a host cell of choice, such as e.g. the yeast species ofchoice. In any case the choice of the terminator is not critical; it maye.g. be from any yeast gene, although terminators may sometimes work iffrom a non-yeast, eukaryotic, gene. Usually a nucleotide sequenceencoding the xylose isomerase comprises a terminator. Preferably, suchterminators are combined with mutations that prevent nonsense mediatedmRNA decay in the host cell of the invention (see for example: Shirleyet al., 2002, Genetics 161:1465-1482).

The transcription termination sequence further preferably comprises apolyadenylation signal.

Optionally, a selectable marker may be present in a nucleic acidconstruct suitable for use in the invention. As used herein, the term“marker” refers to a gene encoding a trait or a phenotype which permitsthe selection of, or the screening for, a host cell containing themarker. The marker gene may be an antibiotic resistance gene whereby theappropriate antibiotic can be used to select for transformed cells fromamong cells that are not transformed. Examples of suitable antibioticresistance markers include e.g. dihydrofolate reductase,hygromycin-B-phosphotransferase, 3′-O-phosphotransferase II (kanamycin,neomycin and G418 resistance). Although the of antibiotic resistancemarkers may be most convenient for the transformation of polyploid hostcells, preferably however, non-antibiotic resistance markers are used,such as auxotrophic markers (URA3, TRPI, LEU2) or the S. pombe TPI gene(described by Russell P R, 1985, Gene 40: 125-130). In a preferredembodiment the host cells transformed with the nucleic acid constructsare marker gene free. Methods for constructing recombinant marker genefree microbial host cells are disclosed in EP-A-O 635 574 and are basedon the use of bidirectional markers such as the A. nidulans amdS(acetamidase) gene or the yeast URA3 and LYS2 genes. Alternatively, ascreenable marker such as Green Fluorescent Protein, lacL, luciferase,chloramphenicol acetyltransferase, beta-glucuronidase may beincorporated into the nucleic acid constructs of the invention allowingto screen for transformed cells.

Optional further elements that may be present in the nucleic acidconstructs suitable for use in the invention include, but are notlimited to, one or more leader sequences, enhancers, integrationfactors, and/or reporter genes, intron sequences, centromers, telomersand/or matrix attachment (MAR) sequences. The nucleic acid constructs ofthe invention may further comprise a sequence for autonomousreplication, such as an ARS sequence.

Preferably, the xylose isomerase is expressed in the cytosol. Cytosolicexpression may be achieved by deletion or modification of amitochondrial or peroxisomal targeting signal.

A cell of the invention may be any suitable cell, such as a prokaryoticcell, such as a bacterium, or a eukaryotic cell. Typically, the cellwill be a eukaryotic cell, for example a yeast or a filamentous fungus.

Yeasts are herein defined as eukaryotic microorganisms and include allspecies of the subdivision Eumycotina (Alexopoulos, C. J., 1962, In:Introductory Mycology, John Wiley & Sons, Inc., New York) thatpredominantly grow in unicellular form.

Yeasts may either grow by budding of a unicellular thallus or may growby fission of the organism. A preferred yeast as a cell of the inventionmay belong to the genera Saccharomyces, Kluyveromyces, Candida, Pichia,Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces or Yarrowia.Preferably the yeast is one capable of anaerobic fermentation, morepreferably one capable of anaerobic alcoholic fermentation.

Filamentous fungi are herein defined as eukaryotic microorganisms thatinclude all filamentous forms of the subdivision Eumycotina. These fungiare characterized by a vegetative mycelium composed of chitin,cellulose, and other complex polysaccharides.

The filamentous fungi of the suitable for use as a cell of the presentinvention are morphologically, physiologically, and genetically distinctfrom yeasts. Filamentous fungal cells may be advantageously used sincemost fungi do not require sterile conditions for propagation and areinsensitive to bacteriophage infections. Vegetative growth byfilamentous fungi is by hyphal elongation and carbon catabolism of mostfilamentous fungi is obligately aerobic. Preferred filamentous fungi asa host cell of the invention may belong to the genus Aspergillus,Trichoderma, Humicola, Acremoniurra, Fusarium or Penicillium. Morepreferably, the filamentous fungal cell may be a Aspergillus niger,Aspergillus oryzae, a Penicillium chrysogenum, or Rhizopus oryzae cell.

Over the years suggestions have been made for the introduction ofvarious organisms for the production of bio-ethanol from crop sugars. Inpractice, however, all major bio-ethanol production processes havecontinued to use the yeasts of the genus Saccharomyces as ethanolproducer. This is due to the many attractive features of Saccharomycesspecies for industrial processes, i. e., a high acid-, ethanol- andosmo-tolerance, capability of anaerobic growth, and of course its highalcoholic fermentative capacity. Preferred yeast species as host cellsinclude S. cerevisiae, S. bulderi, S. barnetti, S. exiguus, S. uvarum,S. diastaticus, K. lactis, K. marxianus or K. fragilis.

A cell of the invention may be able to convert plant biomass,celluloses, hemicelluloses, pectins, rhamnose, galactose, fucose,maltose, maltodextrines, ribose, ribulose, or starch, starchderivatives, sucrose, lactose and glycerol, for example into fermentablesugars. Accordingly, a cell of the invention may express one or moreenzymes such as a cellulase (an endocellulase or an exocellulase), ahemicellulase (an endo- or exo-xylanase or arabinase) necessary for theconversion of cellulose into glucose monomers and hemicellulose intoxylose and arabinose monomers, a pectinase able to convert pectins intoglucuronic acid and galacturonic acid or an amylase to convert starchinto glucose monomers.

A cell of the invention is preferably is a host capable of active orpassive xylose transport into the cell.

Preferably, a cell of the invention:

-   -   is capable of active glycolysis; and/or    -   shows flux through the pentose phosphate pathway; and/or    -   displays xylulose kinase activity so that the xylulose        isomerised from xylose may be metabolised to pyruvate.

The cell further preferably comprises those enzymatic activitiesrequired for conversion of pyruvate to a desired fermentation product,such as ethanol, butanol, lactic acid, 3-hydroxy-propionic acid, acrylicacid, acetic acid, succinic acid, citric acid, fumaric acid, malic acid,itaconic acid, an amino acid, 1,3-propane-diol, ethylene, glycerol, aβ-lactam antibiotic or a cephalosporin.

A preferred cell of the invention is a cell that is naturally capable ofalcoholic fermentation, preferably, anaerobic alcoholic fermentation. Acell of the invention preferably has a high tolerance to ethanol, a hightolerance to low pH (i.e. capable of growth at a pH lower than about 5,about 4, about 3, or about 2.5) and towards organic acids like lacticacid, acetic acid or formic acid and/or sugar degradation products suchas furfural and hydroxy-methylfurfural and/or a high tolerance toelevated temperatures.

Any of the above characteristics or activities of a cell of theinvention may be naturally present in the cell or may be introduced ormodified by genetic modification.

The nucleotide sequence encoding a xylose isomerase is typicallyexpressed or is capable of being expressed in active form in thetransformed host cell. Thus, expression of the nucleotide sequence inthe host cell produces an active xylose isomerase, typically with aspecific activity of at least about 10 U xylose isomerase activity permg protein at about 30° C., preferably at least about 20, at least about25, at least about 30, at least about 50, at least about 100, at leastabout 200, at least about 300, at least about 500, at least about 750 orat least about 1000 U per mg at about 30° C. The specific activity ofthe xylose isomerase expressed in the transformed host cell is hereindefined as the amount of xylose isomerase activity units per mg proteinof cell free lysate of the host cell, e.g. a yeast cell free lysate.Determination of the xylose isomerase activity, amount of protein andpreparation of the cell free lysate are as described herein. Preferably,expression of the nucleotide sequence encoding the xylose isomerase inthe host cell produces a xylose isomerase with a K_(m) for xylose thatis less than 50, 40, 30 or 25 mM, more preferably, the K_(m) for xyloseis about 20 mM or less.

A cell of the invention may comprise one ore more genetic modificationsthat increases the flux of the pentose phosphate pathway. In particular,the genetic modification(s) may lead to an increased flux through thenon-oxidative part pentose phosphate pathway. A genetic modificationthat causes an increased flux of the non-oxidative part of the pentosephosphate pathway is herein understood to mean a modification thatincreases the flux by at least a factor of about 1.1, about 1.2, about1.5, about 2, about 5, about 10 or about 20 as compared to the flux in astrain which is genetically identical except for the geneticmodification causing the increased flux. The flux of the non-oxidativepart of the pentose phosphate pathway may be measured by growing themodified host on xylose as sole carbon source, determining the specificxylose consumption rate and subtracting the specific xylitol productionrate from the specific xylose consumption rate, if any xylitol isproduced. However, the flux of the non-oxidative part of the pentosephosphate pathway is proportional with the growth rate on xylose as solecarbon source, preferably with the anaerobic growth rate on xylose assole carbon source. There is a linear relation between the growth rateon xylose as sole carbon source (μ_(max)) and the flux of thenon-oxidative part of the pentose phosphate pathway. The specific xyloseconsumption rate (Q_(s)) is equal to the growth rate (μ) divided by theyield of biomass on sugar (Y_(xs)) because the yield of biomass on sugaris constant (under a given set of conditions: anaerobic, growth medium,pH, genetic background of the strain, etc.; i.e. Q_(s)=μ/Y_(xs)).Therefore the increased flux of the non-oxidative part of the pentosephosphate pathway may be deduced from the increase in maximum growthrate under these conditions unless transport (uptake is limiting).

One or more genetic modifications that increase the flux of the pentosephosphate pathway may be introduced in the host cell in various ways.These including e.g. achieving higher steady state activity levels ofxylulose kinase and/or one or more of the enzymes of the non-oxidativepart pentose phosphate pathway and/or a reduced steady state level ofunspecific aldose reductase activity. These changes in steady stateactivity levels may be effected by selection of mutants (spontaneous orinduced by chemicals or radiation) and/or by recombinant DNA technologye.g. by overexpression or inactivation, respectively, of genes encodingthe enzymes or factors regulating these genes.

In a preferred host cell, the genetic modification comprisesoverexpression of at least one enzyme of the (non-oxidative part)pentose phosphate pathway. Preferably the enzyme is selected from thegroup consisting of the enzymes encoding for ribulose-5-phosphateisomerase, ribulose-5-phosphate epimerase, transketolase andtransaldolase. Various combinations of enzymes of the (non-oxidativepart) pentose phosphate pathway may be overexpressed. E.g. the enzymesthat are overexpressed may be at least the enzymes ribulose-5-phosphateisomerase and ribulose-5-phosphate epimerase; or at least the enzymesribulose-5-phosphate isomerase and transketolase; or at least theenzymes ribulose-5-phosphate isomerase and transaldolase; or at leastthe enzymes ribulose-5-phosphate epimerase and transketolase; or atleast the enzymes ribulose-5-phosphate epimerase and transaldolase; orat least the enzymes transketolase and transaldolase; or at least theenzymes ribulose-5-phosphate epimerase, transketolase and transaldolase;or at least the enzymes ribulose-5-phosphate isomerase, transketolaseand transaldolase; or at least the enzymes ribulose-5-phosphateisomerase, ribulose-5-phosphate epimerase, and transaldolase; or atleast the enzymes ribulose-5-phosphate isomerase, ribulose-5-phosphateepimerase, and transketolase. In one embodiment of the invention each ofthe enzymes ribulose-5-phosphate isomerase, ribulose-5-phosphateepimerase, transketolase and transaldolase are overexpressed in the hostcell. More preferred is a host cell in which the genetic modificationcomprises at least overexpression of both the enzymes transketolase andtransaldolase as such a host cell is already capable of anaerobic growthon xylose. In fact, under some conditions host cells overexpressing onlythe transketolase and the transaldolase already have the same anaerobicgrowth rate on xylose as do host cells that overexpress all four of theenzymes, i.e. the ribulose-5-phosphate isomerase, ribulose-5-phosphateepimerase, transketolase and transaldolase. Moreover, host cellsoverexpressing both of the enzymes ribulose-5-phosphate isomerase andribulose-5-phosphate epimerase are preferred over host cellsoverexpressing only the isomerase or only the epimerase asoverexpression of only one of these enzymes may produce metabolicimbalances.

The enzyme “ribulose 5-phosphate epimerase” (EC 5.1.3.1) is hereindefined as an enzyme that catalyses the epimerisation of D-xylulose5-phosphate into D-ribulose 5-phosphate and vice versa. The enzyme isalso known as phosphoribulose epimerase; erythrose-4-phosphateisomerase; phosphoketopentose 3-epimerase; xylulose phosphate3-epimerase; phosphoketopentose epimerase; ribulose 5-phosphate3-epimerase; D-ribulose phosphate-3-epimerase; D-ribulose 5-phosphateepimerase; D-ribulose-5-P 3-epimerase; D-xylulose-5-phosphate3-epimerase; pentose-5-phosphate 3-epimerase; or D-ribulose-5-phosphate3-epimerase. A ribulose 5-phosphate epimerase may be further defined byits amino acid sequence. Likewise a ribulose 5-phosphate epimerase maybe defined by a nucleotide sequence encoding the enzyme as well as by anucleotide sequence hybridising to a reference nucleotide sequenceencoding a ribulose 5-phosphate epimerase. The nucleotide sequenceencoding for ribulose 5-phosphate epimerase is herein designated RPE1.

The enzyme “ribulose 5-phosphate isomerase” (EC 5.3.1.6) is hereindefined as an enzyme that catalyses direct isomerisation of D-ribose5-phosphate into D-ribulose 5-phosphate and vice versa. The enzyme isalso known as phosphopentosisomerase; phosphoriboisomerase; ribosephosphate isomerase; 5-phosphoribose isomerase; D-ribose 5-phosphateisomerase; D-ribose-5-phosphate ketol-isomerase; or D-ribose-5-phosphatealdose-ketose-isomerase. A ribulose 5-phosphate isomerase may be furtherdefined by its amino acid sequence. Likewise a ribulose 5-phosphateisomerase may be defined by a nucleotide sequence encoding the enzyme aswell as by a nucleotide sequence hybridising to a reference nucleotidesequence encoding a ribulose 5-phosphate isomerase. The nucleotidesequence encoding for ribulose 5-phosphate isomerase is hereindesignated RPI1.

The enzyme “transketolase” (EC 2.2.1.1) is herein defined as an enzymethat catalyses the reaction: D-ribose 5-phosphate+D-xylulose 5-phosphate<−> sedoheptulose 7-phosphate+D-glyceraldehyde 3-phosphate and viceversa. The enzyme is also known as glycolaldehydetransferase orsedoheptulose-7-phosphate:D-glyceraldehyde-3-phosphateglycolaldehydetransferase. A transketolase may be further defined by itsamino acid. Likewise a transketolase may be defined by a nucleotidesequence encoding the enzyme as well as by a nucleotide sequencehybridising to a reference nucleotide sequence encoding a transketolase.The nucleotide sequence encoding for transketolase is herein designatedTKL1.

The enzyme “transaldolase” (EC 2.2.1.2) is herein defined as an enzymethat catalyses the reaction: sedoheptulose 7-phosphate+D-glyceraldehyde3-phosphate <−> D-erythrose 4-phosphate+D-fructose 6-phosphate and viceversa. The enzyme is also known as dihydroxyacetonetransferase;dihydroxyacetone synthase; formaldehyde transketolase; orsedoheptulose-7-phosphate:D-glyceraldehyde-3-phosphateglyceronetransferase. A transaldolase may be further defined by itsamino acid sequence. Likewise a transaldolase may be defined by anucleotide sequence encoding the enzyme as well as by a nucleotidesequence hybridising to a reference nucleotide sequence encoding atransaldolase. The nucleotide sequence encoding for transketolase fromis herein designated TAL1.

Various means are known to those skilled in the art for expression andoverexpression of enzymes in a cell of the invention. In particular, anenzyme may be overexpressed by increasing the copy number of the genecoding for the enzyme in the host cell, e.g. by integrating additionalcopies of the gene in the host cell's genome, by expressing the genefrom an episomal multicopy expression vector or by introducing aepisomal expression vector that comprises multiple copies of the gene.

Alternatively, overexpression of enzymes in the host cells of theinvention may be achieved by using a promoter that is not native to thesequence coding for the enzyme to be overexpressed, i.e. a promoter thatis heterologous to the coding sequence to which it is operably linked.Although the promoter preferably is heterologous to the coding sequenceto which it is operably linked, it is also preferred that the promoteris homologous, i.e. endogenous to the host cell. Preferably theheterologous promoter is capable of producing a higher steady statelevel of the transcript comprising the coding sequence (or is capable ofproducing more transcript molecules, i.e. mRNA molecules, per unit oftime) than is the promoter that is native to the coding sequence,preferably under conditions where xylose or xylose and glucose areavailable as carbon sources, more preferably as major carbon sources(i.e. more than 50% of the available carbon source consists of xylose orxylose and glucose), most preferably as sole carbon sources. Suitablepromoters in this context include both constitutive and induciblenatural promoters as well as engineered promoters. A preferred promoterfor use in the present invention will in addition be insensitive tocatabolite (glucose) repression and/or will preferably not requirexylose for induction. Promotors having these characteristics are widelyavailable and known to the skilled person. Suitable examples of suchpromoters include e.g. promoters from glycolytic genes, such as thephosphofructokinase (PFK), triose phosphate isomerase (TPI),glyceraldehyde-3-phosphate dehydrogenase (GPD, TDH3 or GAPDH), pyruvatekinase (PYK), phosphoglycerate kinase (PGK) promoters from yeasts orfilamentous fungi; more details about such promoters from yeast may befound in (WO 93/03159). Other useful promoters are ribosomal proteinencoding gene promoters, the lactase gene promoter (LAC4), alcoholdehydrogenase promoters (ADHI, ADH4, and the like), and the enolasepromoter (ENO). Other promoters, both constitutive and inducible, andenhancers or upstream activating sequences will be known to those ofskill in the art. The promoters used in the host cells of the inventionmay be modified, if desired, to affect their control characteristics.

The coding sequence used for overexpression of the enzymes mentionedabove may preferably be homologous to the host cell of the invention.However, coding sequences that are heterologous to the host cell of theinvention may be used.

Overexpression of an enzyme, when referring to the production of theenzyme in a genetically modified host cell, means that the enzyme isproduced at a higher level of specific enzymatic activity as compared tothe unmodified host cell under identical conditions. Usually this meansthat the enzymatically active protein (or proteins in case ofmulti-subunit enzymes) is produced in greater amounts, or rather at ahigher steady state level as compared to the unmodified host cell underidentical conditions. Similarly this usually means that the mRNA codingfor the enzymatically active protein is produced in greater amounts, oragain rather at a higher steady state level as compared to theunmodified host cell under identical conditions. Overexpression of anenzyme is thus preferably determined by measuring the level of theenzyme's specific activity in the host cell using appropriate enzymeassays as described herein. Alternatively, overexpression of the enzymemay be determined indirectly by quantifying the specific steady statelevel of enzyme protein, e.g. using antibodies specific for the enzyme,or by quantifying the specific steady level of the mRNA coding for theenzyme. The latter may particularly be suitable for enzymes of thepentose phosphate pathway for which enzymatic assays are not easilyfeasible as substrates for the enzymes are not commercially available.Preferably in a host cell of the invention, an enzyme to beoverexpressed is overexpressed by at least a factor of about 1.1, about1.2, about 1.5, about 2, about 5, about 10 or about 20 as compared to astrain which is genetically identical except for the geneticmodification causing the overexpression. It is to be understood thatthese levels of overexpression may apply to the steady state level ofthe enzyme's activity, the steady state level of the enzyme's protein aswell as to the steady state level of the transcript coding for theenzyme.

A cell of the invention may comprise one or more genetic modificationsthat increase the specific xylulose kinase activity. Preferably thegenetic modification or modifications causes overexpression of axylulose kinase, e.g. by overexpression of a nucleotide sequenceencoding a xylulose kinase. The gene encoding the xylulose kinase may beendogenous to the host cell or may be a xylulose kinase that isheterologous to the host cell. A nucleotide sequence used foroverexpression of xylulose kinase in the host cell of the invention is anucleotide sequence encoding a polypeptide with xylulose kinaseactivity.

The enzyme “xylulose kinase” (EC 2.7.1.17) is herein defined as anenzyme that catalyses the reaction ATP+D-xylulose=ADP+D-xylulose5-phosphate. The enzyme is also known as a phosphorylating xylulokinase,D-xylulokinase or ATP:D-xylulose 5-phosphotransferase. A xylulose kinaseof the invention may be further defined by its amino acid sequence.Likewise a xylulose kinase may be defined by a nucleotide sequenceencoding the enzyme as well as by a nucleotide sequence hybridising to areference nucleotide sequence encoding a xylulose kinase.

In a cell of the invention, a genetic modification or modifications thatincrease(s) the specific xylulose kinase activity may be combined withany of the modifications increasing the flux of the pentose phosphatepathway as described above. This is not, however, essential.

Thus, a host cell of the invention may comprise only a geneticmodification or modifications that increase the specific xylulose kinaseactivity. The various means available in the art for achieving andanalysing overexpression of a xylulose kinase in the host cells of theinvention are the same as described above for enzymes of the pentosephosphate pathway. Preferably in the host cells of the invention, axylulose kinase to be overexpressed is overexpressed by at least afactor of about 1.1, about 1.2, about 1.5, about 2, about 5, about 10 orabout 20 as compared to a strain which is genetically identical exceptfor the genetic modification(s) causing the overexpression. It is to beunderstood that these levels of overexpression may apply to the steadystate level of the enzyme's activity, the steady state level of theenzyme's protein as well as to the steady state level of the transcriptcoding for the enzyme.

A cell of the invention may comprise one or more genetic modificationsthat reduce unspecific aldose reductase activity in the host cell.Preferably, unspecific aldose reductase activity is reduced in the hostcell by one or more genetic modifications that reduce the expression ofor inactivates a gene encoding an unspecific aldose reductase.Preferably, the genetic modification(s) reduce or inactivate theexpression of each endogenous copy of a gene encoding an unspecificaldose reductase in the host cell. Host cells may comprise multiplecopies of genes encoding unspecific aldose reductases as a result ofdi-, poly- or aneu-ploidy, and/or the host cell may contain severaldifferent (iso)enzymes with aldose reductase activity that differ inamino acid sequence and that are each encoded by a different gene. Alsoin such instances preferably the expression of each gene that encodes anunspecific aldose reductase is reduced or inactivated. Preferably, thegene is inactivated by deletion of at least part of the gene or bydisruption of the gene, whereby in this context the term gene alsoincludes any non-coding sequence up- or down-stream of the codingsequence, the (partial) deletion or inactivation of which results in areduction of expression of unspecific aldose reductase activity in thehost cell.

A nucleotide sequence encoding an aldose reductase whose activity is tobe reduced in the host cell of the invention is a nucleotide sequenceencoding a polypeptide with aldose reductase activity.

In the host cells of the invention, genetic modification that reducesunspecific aldose reductase activity in the host cell may be combinedwith any of the modifications increasing the flux of the pentosephosphate pathway and/or with any of the modifications increasing thespecific xylulose kinase activity in the host cells as described above.This is not, however, essential.

Thus, a host cell of the invention comprising only a geneticmodification or modifications that reduce(s) unspecific aldose reductaseactivity in the host cell is specifically included in the invention.

The enzyme “aldose reductase” (EC 1.1.1.21) is herein defined as anyenzyme that is capable of reducing xylose or xylulose to xylitol. In thecontext of the present invention an aldose reductase may be anyunspecific aldose reductase that is native (endogenous) to a host cellof the invention and that is capable of reducing xylose or xylulose toxylitol. Unspecific aldose reductases catalyse the reaction:aldose+NAD(P)H+H⁺↔alditol+NAD(P)⁺

The enzyme has a wide specificity and is also known as aldose reductase;polyol dehydrogenase (NADP⁺); alditol:NADP oxidoreductase; alditol:NADP⁺1-oxidoreductase; NADPH-aldopentose reductase; or NADPH-aldosereductase.

A particular example of such an unspecific aldose reductase that isendogenous to S. cerevisiae and that is encoded by the GRE3 gene (Traffet al., 2001, Appl. Environ. Microbiol. 67: 5668-74). Thus, an aldosereductase of the invention may be further defined by its amino acidsequence. Likewise an aldose reductase may be defined by the nucleotidesequences encoding the enzyme as well as by a nucleotide sequencehybridising to a reference nucleotide sequence encoding an aldosereductase.

A cell of the invention may be adapted to xylose utilisation byselection of mutants, either spontaneous or induced (e.g. by radiationor chemicals), for growth on xylose, preferably on xylose as sole carbonsource, and more preferably under anaerobic conditions. Selection ofmutants may be performed by techniques including serial passaging ofcultures as e.g. described by Kuyper et al. (2004, FEMS Yeast Res. 4:655-664) or by cultivation under selective pressure in a chemostatculture. In a preferred host cell of the invention at least one of thegenetic modifications described above, including modifications obtainedby selection of mutants, confer to the host cell the ability to grow onxylose as carbon source, preferably as sole carbon source, andpreferably under anaerobic conditions. Preferably the modified host cellproduce essentially no xylitol, e.g. the xylitol produced is below thedetection limit or e.g. less than about 5, about 2, about 1, about 0.5,or about 0.3% of the carbon consumed on a molar basis.

A cell of the invention may have the ability to grow on xylose as solecarbon source at a rate of at least about 0.05, about 0.1, about 0.2,about 0.25 or about 0.3 h⁻¹ under aerobic conditions, or, if applicable,at a rate of at least about 0.03, about 0.05, about 0.07, about 0.08,about 0.09, about 0.1, about 0.12, about 0.15 or about 0.2 h⁻¹ underanaerobic conditions. Preferably the modified host cell has the abilityto grow on a mixture of glucose and xylose (in a 1:1 weight ratio) assole carbon source at a rate of at least about 0.05, about 0.1, about0.2, about 0.25 or about 0.3 h⁻¹ under aerobic conditions, or, ifapplicable, at a rate of at least about 0.03, about 0.05, about 0.1,about 0.12, about 0.15, or about 0.2 h⁻¹ under anaerobic conditions.

A cell of the invention may have a specific xylose consumption rate ofat least about 200, about 250, about 300, about 346, about 350, about400, about 500, about 600, about 750, or about 1000 mg xylose/g cells/h.A cell of the invention may have a yield of fermentation product (suchas ethanol) on xylose that is at least about 40, about 50, about 55,about 60, about 70, about 80, about 85, about 90, about 95 about 98 orabout 99% of the host cell's yield of fermentation product (such asethanol) on glucose. More preferably, the yield of a fermentationproduct (such as ethanol) of a cell of the invention on xylose may beequal to the cell's yield of fermentation product (such as ethanol) onglucose. Likewise, the cell's biomass yield on xylose may be at leastabout 40, about 50, about 55, about 60, about 70, about 80, about 85,about 90, about 95, about 98 or about 99% of the host cell's biomassyield on glucose. More preferably, the cell's biomass yield on xylose,may be equal to the host cell's biomass yield on glucose. It isunderstood that in the comparison of yields on glucose and xylose bothyields are compared under aerobic conditions or both under anaerobicconditions.

A cell of the invention may be capable of using arabinose. A cell of theinvention may, therefore, be capable of converting L-arabinose intoL-ribulose and/or xylulose 5-phosphate and/or into a desiredfermentation product, for example one of those mentioned herein.

Organisms, for example S. cerevisiae strains, able to produce ethanolfrom L-arabinose may be produced by modifying a cell introducing thearaA (L-arabinose isomerase), araB (L-ribulokinase) and araD(L-ribulose-5-P4-epimerase) genes from a suitable source. Such genes maybe introduced into a cell of the invention is order that it is capableof using arabinose. Such an approach is described in WO2003/095627.

A cell of the invention may be a cell suitable for the production ofethanol. A cell of the invention may, however, be suitable for theproduction of fermentation products other than ethanol. Suchnon-ethanolic fermentation products include in principle any bulk orfine chemical that is producible by a eukaryotic microorganism such as ayeast or a filamentous fungus.

Such fermentation products may be, for example, butanol, lactic acid,3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid,citric acid, malic acid, fumaric acid, itaconic acid, an amino acid,1,3-propane-diol, ethylene, glycerol, a β-lactam antibiotic or acephalosporin. A preferred modified host cell of the invention forproduction of non-ethanolic fermentation products is a host cell thatcontains a genetic modification that results in decreased alcoholdehydrogenase activity.

In a further aspect the invention relates to fermentation processes inwhich the modified host cells of the invention are used for thefermentation of a carbon source comprising a source of xylose, such asxylose. In addition to a source of xylose the carbon source in thefermentation medium may also comprise a source of glucose. The source ofxylose or glucose may be xylose or glucose as such or may be anycarbohydrate oligo- or polymer comprising xylose or glucose units, suchas e.g. lignocellulose, xylans, cellulose, starch and the like. Forrelease of xylose or glucose units from such carbohydrates, appropriatecarbohydrases (such as xylanases, glucanases, amylases and the like) maybe added to the fermentation medium or may be produced by the modifiedhost cell. In the latter case the modified host cell may be geneticallyengineered to produce and excrete such carbohydrases. An additionaladvantage of using oligo- or polymeric sources of glucose is that itenables to maintain a low(er) concentration of free glucose during thefermentation, e.g. by using rate-limiting amounts of the carbohydrases.This, in turn, will prevent repression of systems required formetabolism and transport of non-glucose sugars such as xylose.

In a preferred process the modified host cell ferments both the xyloseand glucose, preferably simultaneously in which case preferably amodified host cell is used which is insensitive to glucose repression toprevent diauxic growth. In addition to a source of xylose (and glucose)as carbon source, the fermentation medium will further comprise theappropriate ingredient required for growth of the modified host cell.Compositions of fermentation media for growth of microorganisms such asyeasts are well known in the art. The fermentation process is a processfor the production of a fermentation product such as e.g. ethanol,butanol, lactic acid, 3-hydroxy-propionic acid, acrylic acid, aceticacid, succinic acid, citric acid, malic acid, fumaric acid, itaconicacid, an amino acid, 1,3-propane-diol, ethylene, glycerol, a β-lactamantibiotic, such as Penicillin G or Penicillin V and fermentativederivatives thereof, and a cephalosporin.

The fermentation process may be an aerobic or an anaerobic fermentationprocess. An anaerobic fermentation process is herein defined as afermentation process run in the absence of oxygen or in whichsubstantially no oxygen is consumed, preferably less than about 5, about2.5 or about 1 mmol/L/h, more preferably 0 mmol/L/h is consumed (i.e.oxygen consumption is not detectable), and wherein organic moleculesserve as both electron donor and electron acceptors. In the absence ofoxygen, NADH produced in glycolysis and biomass formation, cannot beoxidised by oxidative phosphorylation. To solve this problem manymicroorganisms use pyruvate or one of its derivatives as an electron andhydrogen acceptor thereby regenerating NAD⁺.

Thus, in a preferred anaerobic fermentation process pyruvate is used asan electron (and hydrogen acceptor) and is reduced to fermentationproducts such as ethanol, butanol, lactic acid, 3-hydroxy-propionicacid, acrylic acid, acetic acid, succinic acid, citric acid, malic acid,fumaric acid, an amino acid, 1,3-propane-diol, ethylene, glycerol, aβ-lactam antibiotic and a cephalosporin.

The fermentation process is preferably run at a temperature that isoptimal for the modified host cell. Thus, for most yeasts or fungal hostcells, the fermentation process is performed at a temperature which isless than about 42° C., preferably less than about 38° C. For yeast orfilamentous fungal host cells, the fermentation process is preferablyperformed at a temperature which is lower than about 35, about 33, about30 or about 28° C. and at a temperature which is higher than about 20,about 22, or about 25° C.

A preferred process is a process for the production of a ethanol,whereby the process comprises the steps of: (a) fermenting a mediumcontaining a source of xylose with a modified host cell as definedabove, whereby the host cell ferments xylose to ethanol; and optionally,(b) recovery of the ethanol. The fermentation medium may also comprise asource of glucose that is also fermented to ethanol. In the process thevolumetric ethanol productivity is preferably at least about 0.5, about1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 5.0 or about 10.0g ethanol per liter per hour. The ethanol yield on xylose and/or glucosein the process preferably is at least about 50, about 60, about 70,about 80, about 90, about 95 or about 98%. The ethanol yield is hereindefined as a percentage of the theoretical maximum yield.

The invention also relates to a process for producing a fermentationproduct, such as a product selected from the group consisting of butanollactic acid, 3-hydroxy-propionic acid, acrylic acid, acetic acid,succinic acid, citric acid, malic acid, fumaric acid, itaconic acid, anamino acid, 1,3-propane-diol, ethylene, glycerol, a β-lactam antibioticand a cephalosporin. The process preferably comprises fermenting amedium containing a source of xylose with a modified host cell asdefined herein above, whereby the host cell ferments xylose to thefermentation product.

The invention also provides a process for producing a fermentationproduct, such as a product selected from the group consisting ofethanol, butanol, lactic acid, 3-hydroxy-propionic acid, acrylic acid,acetic acid, succinic acid, citric acid, malic acid, fumaric acid,itaconic acid, an amino acid, 1,3-propane-diol, ethylene, glycerol, aβ-lactam antibiotic and a cephalosporin. The process preferablycomprises fermenting a medium containing at least a source of xylose anda source of L-arabinose with a cell as defined above which is able touse both of xylose and L-arabinose such that the cell ferments xyloseand L-arabinose to the fermentation product.

The invention also provides a process for producing a fermentationproduct, such as a product selected from the group consisting ofethanol, butanol, lactic acid, 3-hydroxy-propionic acid, acrylic acid,acetic acid, succinic acid, citric acid, malic acid, fumaric acid,itaconic acid, an amino acid, 1,3-propane-diol, ethylene, glycerol, aβ-lactam antibiotic and a cephalosporin. The process preferablycomprises fermenting a medium containing at least a source of xylose anda source of L-arabinose with a cell as defined above and a cell able touse L-arabinose, whereby each cell ferments xylose and/or arabinose tothe fermentation product.

A process of the invention may also comprise recovery of thefermentation product. The medium with which the process is carried outmay also contain a source of glucose.

The process according to the present invention may be run under aerobicand anaerobic conditions. Preferably, the process is carried out undermicro-aerophilic or oxygen limited conditions.

An anaerobic fermentation process is herein defined as a fermentationprocess run in the absence of oxygen or in which substantially no oxygenis consumed, preferably less than about 5, about 2.5 or about 1mmol/L/h, and wherein organic molecules serve as both electron donor andelectron acceptors.

An oxygen-limited fermentation process is a process in which the oxygenconsumption is limited by the oxygen transfer from the gas to theliquid. The degree of oxygen limitation is determined by the amount andcomposition of the ingoing gasflow as well as the actual mixing/masstransfer properties of the fermentation equipment used. Preferably, in aprocess under oxygen-limited conditions, the rate of oxygen consumptionis at least about 5.5, more preferably at least about 6, such as atleast 7 mmol/L/h.

The following Examples illustrate the invention:

EXAMPLES

General Molecular Biology Techniques

Unless indicated otherwise, the methods used are standard biochemicaltechniques. Examples of suitable general methodology textbooks includeSambrook et al., Molecular Cloning, a Laboratory Manual (1989) andAusubel et al., Current Protocols in Molecular Biology (1995), JohnWiley & Sons, Inc.

Growth experiments were performed using either Verduyn medium (Verduyn,1992) or YEPh-medium (10 g/l yeast extract, 20 g/l phytone),supplemented with sugars as indicated in the examples. For solid YEPhmedium, 20 g/l agar was added to the liquid medium prior tosterilization.

Transformation of Yeast Cells with Circular DNA

Yeast transformation was done according to the method described by Chenet al (Current Genetics (1992), Volume 21, Number 1, 83-84) in case ofplasmid DNA.

Transformation of Yeast Cells with Linear DNA Fragments byElectroporation

Yeast cells were cultured by inoculating 25 ml of YEPh-medium containing2% glucose with a single yeast colony. The flask was incubated overnightat 30° C., 280 rpm.

The optical density at 600 nm was determined and the amount needed toobtain an optical density of 0.2 was transferred to 100 ml YEPh-mediumwith 2% glucose. The cells were grown for 4 to 5 hours at 30° C., 280rpm, in order to reach an optical density of approximately 1.2 to 1.3,which corresponds to 2 to 3 generations. Cells were collected bycentrifugation and resuspended in 28 ml TE (10 mM Tris.HCl, 1 mM EDTA,pH 7.5). 3 ml of a 1 M LiAC solution (set at pH 7.5 with diluted HAc)was added. The cells were gently shaken in a rotary incubator (150 rpm,30° C.) for 45 minutes. After addition of 500 μl of a 1 M DTT(dithiothreitol) solution, the cells were incubated once more underthese conditions, for 15 minutes. The volume was made up to 100 ml withsterile, ice-cold milliQ water. The cells were collected bycentrifugation.

The supernatant was discarded and the pelleted cells were washed with 50ml of sterile, ice cold milliQ water, and collected by centrifugation. Asubsequent washing treatment was done with 30 ml of an ice cold 1 Msorbitol solution. After centrifugation, the supernatant was discardedand the cell pellet was resuspended in 4 ml of an ice cold 1 M sorbitolsolution. After centrifugation, the supernatant was discarded and thecell pellet was resuspended in 300 μl of an ice cold 1 M sorbitolsolution.

For each transformation, 40 μl of the cell suspension was transferredinto ice cold Eppendorf tubes. The transforming DNA and 5 μg salmonsperm DNA (as carrier DNA) was added, together in a maximum volume of 20μl. The DNA should be dissolved in TE. The Eppendorf tube was carefullytapped in order to mix the content gently. Subsequently, the content wastransferred to a pre-chilled (on ice) electroporation cuvette with a gapof 0.2 cm and a pulse (using e.g. a BioRad Electroporation Device) at1.5 kV, 200 Ohm and 25 μF was applied. The pulse time should be around 5ms.

The cells were immediately transferred to 200 μl 1 M sorbitol.Subsequently, 4 ml of YEPh 2% glucose was added and the cell suspensionwas incubated at 30° C. for 1 hour. After this hour, the cells werecollected by centrifugation, the supernatant was discarded and thepellet resuspended in 4 ml of 1 M sorbitol. The cells were againcollected by centrifugation, the supernatant was discarded and thepellet resuspended in 300 μl of 1 M sorbitol. The cells were diluted asappropriate and used in selective media.

Ethanol Production

Pre-cultures were prepared by inoculating 25 ml Verduyn-medium (Verduynet al., Yeast 8:501-517, 1992) supplemented with 2% glucose in a 100 mlshake flask with a frozen stock culture or a single colony from an agarplate. After incubation at 30° C. in an orbital shaker (280 rpm) forapproximately 24 hours, this culture was harvested and used fordetermination of carbon dioxide evolution and ethanol productionexperiments.

Cultivations for ethanol production were performed at 30° C. in 100 mlsynthetic model medium (Verduyn-medium (Verduyn et al., Yeast 8:501-517,1992) with 5% glucose, 5% xylose, 3.5% arabinose, 1% galactose and 0.5%mannose) in the BAM (Biological Activity Monitor, Halotec, theNetherlands). The pH of the medium was adjusted to 4.2 with 2 MNaOH/H₂SO₄ prior to sterilisation. The synthetic medium for anaerobiccultivation was supplemented with 0.01 g/l ergosterol and 0.42 g/l Tween80 dissolved in ethanol (Andreasen and Stier. J. Cell Physiol. 41:23-36,1953; and Andreasen and Stier. J. Cell Physiol. 43:271-281, 1954). Themedium was inoculated at an initial OD⁶⁰⁰ of approximately 2. Cultureswere stirred by a magnetic stirrer. Anaerobic conditions developedrapidly during fermentation as the cultures were not aerated. Carbondioxide production was monitored constantly. Sugar conversion andproduct formation (ethanol, glycerol) was analyzed by NMR. Growth wasmonitored by following optical density of the culture at 600 nm on a LKBUltrospec K spectrophotometer.

Example 1 Construction of Xylose Isomerase Expression Vector

A synthetic codon-pair optimized xylA gene was designed based on theprimary amino acid sequence and synthesized by GeneArt (Regensburg,Germany). Codon-pair optimization was performed as described previously(WO2008000632). The nucleotide sequence is included in here as SEQ ID NO1, the corresponding amino acid sequence as SEQ ID NO 2.

The open reading frame was cloned into the yeast shuttle vector p427-TEF(FIG. 1; DualSystems Biotech, Schlieren, Switzerland) using therestriction enzymes SpeI and XmaI.

The plasmid was called pPWT215.

Example 2 Transformation of BIE104P1 with pPWT215

A S. cerevisiae strain, BIE104P1 (MATa URA3 HIS3 LEU2 TRP1 MAL2-8 SUC2GRE3::[TPI1p-TAL1_ADH1p-TKL1_PGI1p-RPE1_ENO1p-RKI1]) as disclosed inWO2009109633, in which the GRE3-gene was replaced by the genes of thenon-oxidative part of the pentose phosphate pathway was transformed withthe plasmid pPWT215, using the One-Step Yeast Transformation methoddescribed by Chen et al (1992). As a negative control, milliQ was used.The final pellet of the transformation procedure was resuspended in 1 mlof YEPh 2% glucose (see above). 50 μl of this transformation mixture wasplated on a YEPh agar plate supplemented with 2% glucose and 200 μgG418/ml. The remaining 950 μl was transferred to a 100 ml shake flaskcontaining 25 ml Verduyn medium, supplemented with 2% xylose, 10 μgG418/ml and 250 μl of a Pen-Strep solution (Gibco/Invitrogen). Theinoculated flasks (mock transformation and plasmid transformation) wereincubated in a rotary shaker at 30° C. and 280 rpm. The optical densitywas followed in time.

Example 3 Growth Experiment

The progression of the growth experiment following transformation isshown in FIG. 2.

During the first ten days of the growth experiment, the optical densityat 600 nm of the cultures hardly increased. After 20 days (indicatedwith “1” in FIG. 2) the optical density of the plasmid transformed cellculture reached a value larger than 20. Subsequently, an amount of theculture was transferred to a fresh aliquot of 25 ml Verduyn mediumcontaining 2% xylose, 10 μg G418/ml and 250 μl Pen-Strep. As is clearfrom FIG. 2, 4 transfers were made. The growth experiment was performedin the presence of air, except for the last culture, after the fourthtransfer, which was performed in a shake flask with a waterlock(anaerobic conditions).

From FIG. 2 it can be concluded that yeast cells of strain BIE104P1transformed with pPWT215 have gained the ability to utilize xylose as asole carbon and energy source, while yeast cells of the same strain,mock transformed, did not show an increase in optical density, hencecannot grow on xylose.

Example 4 Construction of a Pentose Fermenting Cell

S. cerevisae strain BIE292 is a derivative of strain BIE201. Theconstruction of this strain has been disclosed in PCT/EP2011/056242,example 7. It is the result of a back-cross of strain BIE201, a strainwhich is optimized by transformation and adaptive evolution forefficient arabinose fermentation, and its parent, prior to adaptiveevolution, strain BIE104A2P1a. Strain BIE292 expresses the genes araA,araB and araD from Lactobacillus plantarum and the non-oxidative pentosephosphate pathway genes TAL1, TKL1, RPE1 and RKI1 constitutively and theGRE3 gene encoding aldose reductase has been deleted. In addition,BIE292 has an amplification of this arabinose cassette on chromosome VIIand the SNP in the GAL80 gene (nucleotide change A436C, where the A ofthe startcodon ATG is 1).

Subsequently, strain BIE292 was transformed with PCR fragments of thecodon-pair optimized xylA gene, including promoter and terminatorsequences, flanked with 100 bp overlapping regions to Ty1 sequences inthe genome. The xylA expression cassette was amplified by PCR usingpPWT215 as a template. The PCR reaction was performed with the primersequences SEQ ID NO 3 and SEQ ID NO 4 using Phusion DNA polymerase(Finnzymes) using the instructions of the supplier. The amplifiedexpression cassette was ethanol precipitated and stored at −20° C. priorto use. The precipitated DNA was collected by centrifugation and the DNApellet was washed with 70% ethanol and subsequently air dried. The DNAwas dissolved at a concentration of approximately 1 μg of DNA/μl inTE-buffer.

Yeast strain BIE292 was grown in YEPh-medium containing 2% glucose.Competent cells were prepared for electroporation, as described above.Electrocompetent cells were transformed with 20 μg PCR product. As anegative control, milliQ water was used.

Transformation mixes of both the xylA- and mock transformation weredirectly transferred to 4 different shake flasks each containing 25 mlVerduyn medium supplemented with 2% xylose and 250 μl pen/strep. As nodominant selection marker is used, selection of correct transformants onplate after transformation was not performed.

The eight shake flasks were incubated in a rotary shaker at 30° C. and280 rpm. Growth was monitored by measuring the optical density at 600 nmfrequently.

Example 5 Selection and Characterization of a Pentose Fermenting YeastStrain

If the culture described in Example 4 has reached an optical density at600 nm greater than 15, an aliquot of 250 μl is transferred to a flaskcontaining Verduyn medium supplemented with 2% xylose. The opticaldensity at 600 nm is monitored frequently. The growth rate is determinedwith the use of the optical density data.

After several cycles of inoculation on xylose containing media, Verduynmedium containing 2% arabinose as a carbon source is being used, inorder to keep the selective pressure on arabinose utilization. Likewise,a flask containing Verduyn medium supplemented with a mixture of hexoses(i.e. glucose, galactose and/or mannose) and pentoses (i.e. arabinoseand/or xylose) is used. The culturing experiments are initiallyperformed under aerobic conditions, but are subsequently performed underanaerobic conditions, for instance when the growth rate exceeds a valueof about 0.07 per hour or higher, on both arabinose and xylose.

After several cycles of growth and re-inoculation under anaerobicconditions, an aliquot of the cultures is diluted to about 100-1000colony forming units (CFU) per milliliter and subsequently aliquots of10-100 μl are plated out on YEPh-agar plates containing 2% glucose.Plates are incubated for at least 48 hours at 30° C., or until singlecolonies are visible.

Single colony isolates are tested in the BAM (vide supra). Single colonyisolates are selected on basis of the ability to ferment all five sugarsefficiently, as is inferred from the carbon dioxide profile and the NMRdata of the sugars, ethanol and by-products.

The best strains are characterized by genetic and molecular biologytechniques known to those skilled in the art, such as PCR, Southernblot(s), FIGE and resequencing.

Example 6 Selection and Characterization of a Pentose Fermenting YeastStrain

When the culture described in Example 4 had reached an optical densityat 600 nm greater than 10, an aliquot of 25 μl was transferred to ashake flask containing Verduyn medium supplemented with 2% xylose. Theoptical density was monitored frequently and the growth rate wasdetermined with the use of these data. When the culture reached anoptical density at 600 nm greater than 15, and aliquot of the culturewas transferred to a shake flask containing Verduyn medium supplementedwith 2% xylose and the shake flask was closed with a water lock enablinganaerobic culturing. From this point, incubations were performed in anorbital shaker at 30° C. and 100 rpm. Growth of the culture wasmonitored by measuring the optical density at 600 nm and the growth ratewas calculated based on these data. When the optical density at 600 nmof the culture reached a value greater than 3.75, an aliquot of theculture was transferred to a shake flask containing Verduyn mediumsupplemented with 2% arabinose. The shake flask was closed with a waterlock. In case the culture reached an optical density at 600 nm with avalue greater than 5, an aliquot of the culture was again transferred toa shake flask containing Verduyn medium supplemented with 2% xylose andthis shake flask was closed with a water lock. A typical result of thisgrowth experiment is given in FIG. 3.

As is clear from FIG. 3, transformation of BIE292 with XyIA fromClostridium beyerinckii enabled this strain to rapidly obtain axylose-consuming phenotype, while the mock transformation did not resultin xylose-consuming cells.

Table 1 presents the growth rates of S. cerevisiae strains transformedwith xylose isomerase genes from several organisms. Strain BIE292XI (C.beyerinckii) demonstrated the highest growth rate under aerobicconditions upon initial growth on xylose as sole carbon source aftertransformation. In addition, BIE292XI (C. beyerinckii) was the onlystrain able to grow on xylose under anaerobic conditions withoutextensive evolutionary engineering.

TABLE 1 Growth rates of BIE292XI(C. beyerinckii) and publicallyavailable data of a Saccharomyces cerevisiae strains transformed with XIof Clostridium phytofermentans (ref. 1), Orpinomyces (ref. 2), Piromyces(RWB202) (ref. 3), and Thermus thermophilus (ref. 4), upon initialgrowth on xylose. Xylose isomerase Aerobic Anaerobic C. beyerincki 0.061h⁻¹ 0.056 h⁻¹ C. phytofermentans 0.039 h⁻¹ — Orpinomyces  0.01 h⁻¹ —Piromyces 0.005 h⁻¹ — T. thermophilus — —

The data presented in the table above are all obtained from differentstrain backgrounds.—indicates not measured or not tested. For the S.cerevisiae strain harboring XI from T. Thermophilus it is noted thatconsumption of xylose was observed, but consumption levels were too lowto support growth.

The invention claimed is:
 1. A yeast cell comprising a heterologousnucleotide sequence encoding a polypeptide comprising at least 95%sequence identity to the amino acid sequence of SEQ ID NO: 2 and havingxylose isomerase activity, wherein said yeast cell can grow on xylose asa sole carbon source under aerobic and anaerobic conditions.
 2. Theyeast cell of claim 1, wherein said heterologous nucleotide sequenceencoding said polypeptide having polypeptide having xylose isomeraseactivity is obtained from a cell of the genus Clostridium.
 3. The yeastcell of claim 1, wherein said yeast cell is of the genus Saccharomyces,Kluyveromcyes, Candida, Pichia, Schizosaccharomyces, Hansenula,Klockera, Schwanniomyces, or Yarrowia.
 4. The yeast cell of claim 3,wherein said yeast cell is of the species S. cerevisiae, S. bulderi, S.barnetti, S. exiguus, S. uvarum, S. diastaticus, K. lactis, K.marxianus, or K. fragilis.
 5. The yeast cell of claim 1, wherein saidyeast cell further comprises at least one genetic modification resultingin: a. an increase in transport of xylose in the cell; b. an increase inxylulose kinase activity; c. an increase in flux through the pentosephosphate pathway; d. a decrease in aldose reductase activity; e. adecrease in sensitivity to catabolite repression; f. an increase intolerance to ethanol, osmolarity or organic acids; or g. a reducedproduction of by-products, as compared with a yeast cell without said atleast one genetic modification, wherein said at least one geneticmodification is selected from the group consisting of overexpression ofat least one gene encoding an enzyme of the non-oxidative part of thepentose phosphate pathway, overexpression of a gene encoding a xylulosekinase, expression of genes araA, araB and araD from Lactobacillusplantarum, inactivation of a gene encoding an endogenous unspecificaldose reductase, and a combination thereof.
 6. The yeast cell of claim5, wherein said at least one genetic modification is overexpression ofat least one gene encoding an enzyme of the non-oxidative part of thepentose phosphate pathway.
 7. The yeast cell of claim 6, wherein saidgene ene encodes a ribulose-5-phosphate isomerase, aribulose-5-phosphate epimerase, a transketolase, and/or a transaldolase.8. The yeast cell of claim 5, wherein said at least one geneticmodification is overexpression of a gene encoding a xylulose kinase. 9.The yeast cell of claim 5, wherein said at least one geneticmodification is inactivation of a gene encoding an endogenous unspecificaldose reductase.
 10. The yeast cell of claim 1, wherein said yeast cellfurther comprises overexpression of TAL1, TKL1, RPE1, and RKI1 and theability to use L-arabinose.
 11. The yeast cell of claim 5, wherein genesaraA, araB and araD from Lactobacillus plantarum are expressed.
 12. Theyeast cell of claim 5, comprising at least one constitutively expressedor constitutively overexpressed gene stably integrated into the genomeof said yeast cell.
 13. A process for producing a fermentation product,comprising: fermenting a medium containing xylose in the presence of theyeast cell of claim 1, under conditions whereby said fermentationproduct is produced.
 14. The process of claim 13, wherein saidfermentation product is ethanol, butanol, lactic acid,2-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid,citric acid, malic acid, fumaric acid, itaconic acid, an amino acid,1,3-propane-diol, ethylene, glycerol, a β-lactam antibiotic and/or acephalosporin.
 15. The process of claim 13, wherein to said process isanaerobic.